A STUDY OF EXPERIMENTAL REINFECTION BY TRYPANOSOMA CRUZI IN DOGS

Transcription

1 Am. J. Trop. Med. Hyg., 65(6), 2001, pp Copyright 2001 by The American Society of Tropical Medicine and Hygiene A STUDY OF EXPERIMENTAL REINFECTION BY TRYPANOSOMA CRUZI IN DOGS EVANDRO M. M. MACHADO, ALEXANDRE J. FERNANDES, SILVANE M. F. MURTA, RICARDO W. A. VITOR, DEOLINO J. CAMILO JÚNIOR, SIMONE W. PINHEIRO, EDISON REIS LOPES, SHEILA J. ADAD, ALVARO J. ROMANHA, AND JOÃO CARLOS PINTO DIAS Centro de Pesquisas René Rachou, Fiocruz, Belo Horizonte, Minas Gerais, Brazil; Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; Faculdade de Medicina do Triângulo Mineiro, Uberaba, Minas Gerais, Brazil Abstract. The role of reinfection in the evolution of Chagas disease was evaluated in dogs alternately infected with the and strains of Trypanosoma cruzi. A parasitologic, serologic, clinical, and electrocardiographic follow-up was carried out on the infected and noninfected dogs. The dogs were reinfected five times over a period of 38 months. No deaths were observed during the experiment. They presented a brief oligosymptomatic acute phase. The level of parasitemia decreased progressively with the number of reinfections. Bloodstream parasites were not detectable after the fifth reinfection. All parasite samples isolated during the follow-up were zymodeme B, corresponding to strain, irrespective of the strain with which the dogs were first infected and of the triatomine species used for isolation. Conversely, amplification by the polymerase chain reaction of a segment of the T. cruzi mini-exon gene showed the simultaneous presence of both strains in three of the eight reinfected animals. Antibody titers were greater among the dogs successively infected than those infected only once. Neither amastigotes nor T. cruzi DNA were detected in the tissues of the infected dogs. Alterations related to Chagas disease were identified only in the heart and consisted of chronic focal and discrete myocarditis, compatible with the indeterminate form of Chagas disease. All infected dogs developed this form of the disease, which was independent of the number of infections. INTRODUCTION Chagas disease is a trypanosomiasis found in the Americas whose etiological agent is Trypanosoma cruzi. It is estimated that 90 million individuals are at risk of infection, million are infected with T. cruzi, and 50,000 patients die each year of this disease. 1 There are millions of cases in which the disease follows an implacable and progressive evolution. The different epidemiologic and clinical manifestations of the infection and the mechanisms and/or factors that determine the development of morbidity are still unclear. Several important points remain to be clarified with regard to the natural history of Chagas disease. One of these is related to the role of reinfections in the developmental potential of the parasite. This has an important role in the prophylaxis and therapy of this disease. Several studies have investigated this aspect 2 12 without being able to resolve this question. The dog has been extensively used as an experimental model in the study of Chagas disease, since it has characteristic aspects of the infection in humans. In this work, we studied the course of T. cruzi infection in dogs that had been experimentally reinoculated with heterologous and homologous strains from parasitologic, serologic, and anatomo-pathologic viewpoints. In addition, we studied the dynamics of evolution of the strains of T. cruzi inoculated by means of the isoenzyme profiles and variability of the mini-exon gene of the parasite. MATERIALS AND METHODS Trypanosoma cruzi strains. Two Brazilian strains were used in the study. The first strain was, isolated by hemoculture of a patient from Bambuí, Minas Gerais, who had chronic Chagas disease involving T. cruzi zymodeme B (ZB), characteristic of strains circulating in the domiciliary environment (Romanha AJ, unpublished data). The second strain was, isolated from a naturally-infected triatomine (Panstrongylus megistus) from the state of Santa Catarina, 958 belonging to zymodeme 1 (Z1), characteristic of strains that circulate in the sylvatic environment. 16 Animals and inoculum. Sixteen cross-bred dogs (4 months old) of both sexes, originating from two litters, were used in the study. The animals were maintained in individual kennels in the animal house of the Posto Avançado de Estudos Emmanuel Dias, Bambui (Minas Gerais) according to the code of ethics of the Colégio Brasileiro de Experimentação Animal (COBEA). Twelve dogs were inoculated or reinoculated intraperitoneally with 10 3 blood trypomastigotes/kg of body weight and subdivided into four groups, as shown in Table 1, and two noninfected dogs belonging to Group E. Eight dogs were subjected to five reinfections. The animals were follow-up for a period of 38 months; the last infection was carried out in the 28th month. Examinations of fresh blood were performed daily from the fifth day until the 60th day after all infections for the detection of the parasites. The parasitemia was quantified according to the technique of Brener. 17 Xenodiagnosis and re-isolation of the parasites. Xenodiagnosis was also used to monitor parasitemia. This was done on the seventh day after the initial infection, and was repeated at two-week intervals during the first month and at monthly intervals thereafter for a period of 38 months. This procedure was repeated in all dogs in the experimental groups. Ten third-instar nymphs of Triatoma infestans, P. megistus, and Rhodnius neglectus and first-instar nymphs of Dipetalogaster maximus were used (a total of 40 nymphs per xenodiagnostic test). The insects were examined individually by abdominal compression 30 days after their blood meal. The intestinal contents obtained was diluted in phosphate-buffered saline, ph 7.2, placed on a slide under a mm coverslip, and all fields were examined by optical microscopy (magnification 100 ). Parasitemia was evaluated by xenodiagnosis to calculate the percentage of positive triatomines (number of positive insects/total number of insects examined 100). The parasites were isolated by xen-

2 REINFECTION BY T. CRUZI IN DOGS 959 TABLE 1 Groups of dogs infected and reinfected with Trypanosoma cruzi Group (n) Infected* A(2) B(2) Reinfected* C(4) D(4) Chronogram of inoculation of T. cruzi strains (month) * The dogs were inoculated intraperitoneally with 10 3 blood trypomastigotes/kg of body weight. oculture from triatomines used in the xenodiagnoses 18 to evaluate the co-existing populations. Serologic tests. An indirect immunofluoresence (IIF) test and an enzyme-linked immunosorbent assay (ELISA) were performed to detect IgG antibodies to T. cruzi as described. 19 Blood was obtained monthly by puncture of the femoral vein. Serum was separated by centrifugation at 1,800 g for 10 min at room temperature, aliquoted, and stored at 20 C. The antigens used in both techniques were obtained from the Y strain of T. cruzi cultivated in liver infusion tryptose (LIT) medium. For the IIF test, the sera were successively diluted from 1:10 to 1:640 and antibody was detected by adding fluorescent-labeled anti-dog IgG (Sigma, St. Louis, MO). For the ELISA, the sera were diluted to 1: 320 and peroxidase-conjugated anti-dog IgG (Sigma) was used as the secondary antibody. The results were read at an absorbance of 490 nm. Molecular characterization of the inoculated strains. Isoenzymatic profile. Parasites isolated by xenoculture that were in exponential growth in LIT medium at 28 C (approximately 10 9 cells/ml) were washed three times in buffer solution (Krebs-Ringer-Tris, ph 7.2), followed by centrifugation at 1,000 g for 10 min at 4 C. The pellet obtained was stored at 70 C until used in the preparation of enzymatic extracts and DNA. Sediments containing parasites were thawed and subjected to osmotic lysis in an enzymatic stabilizer at 4 C at a ratio of 1:1. The lysate was centrifuged at 15,000 g for1hrat4 C and the supernatant was cryopreserved in liquid nitrogen. 20 The isoenzymes were separated by electrophoresis in a horizontal thin-layer starch gel refrigerated at 4 C. The six enzymes used in the characterization of the isolated samples were alanine aminotranferase (ALT) (E.C ), aspartate aminotransferase (AST) (E.C ), glucose 6-phosphate dehydrogenase (G6PD) (E.C ), glucose phosphate isomerase (GPI) (E.C ), malate dehydrogenase (MDH) (E.C ), and phosphoglycomutase (PGM) (E.C ). Variability of the mini-exon gene. The preparation of DNA involved extraction from parasite cultures using a Wizard DNA purification kit (Promega, Madison, WI). Molecular typing was based on amplification of part of the intergenic region of the mini-exon gene of T. cruzi. 21 The amplified products were analyzed by electrophoresis in polyacrylamide gels and stained with silver. 22 Clinical evaluation. Electrocardiograms were evaluated using nine derivations (D1, D2, D3, avr, avl, avf, V1, V3, V5) with a speed of 50 mm/sec. Prior to the first infection, two electrocardiograms were made per animal at an interval of two days to obtain normal electrocardiographic patterns. Two examinations were performed between the first and second inoculations. The rectal temperatures of all the dogs were checked daily and each animal was weighed every two weeks. Anatomo-pathologic examination. The animals were killed according to the code of ethics of COBEA at the chronic phase (38th month) of infection. A complete necroscopic study was made of nine infected or reinfected dogs and four noninfected dogs, in which signs of inflammation and/or possible visceromegalia were evaluated. Fragments of heart, esophagus, small and large intestines, nervous system, liver, spleen, kidneys, adrenal glands, and genital organs were fixed in 4% formaldehyde and processed after embedding in paraffin. Histologic sections (7 m thick) were stained with hematoxylin and eosin. Four blocks were made from heart tissue, representing all chambers. In addition to staining with hematoxylin and eosin, we also used Masson s trichromic technique (one section per block) and peroxidaseantiperoxidase (PAP) for T. cruzi (five sections from each block giving a total of 20 sections). The slides were evaluated with regard to the presence and intensity of the inflammatory process, and examined for amastigote forms of T. cruzi. Detection of parasite DNA by the polymerase chain reaction (PCR). Fragments of all chambers of the heart were frozen at 70 C to detect parasite DNA by PCR. The DNA was isolated according to the procedure of Passos and others. 23 A repetitive sequence of 195 basepairs of nuclear DNA of T. cruzi was used as a PCR amplification target using the primers 5 -CGA GCT CTT GCC CAC ACG GGT GCT-3 and 5 -CCT CCA AGC AGC GGA TAG TTC AGG The reagents used for amplification were PCR buffer (10 mm Tris-HCl, ph 8.0, 50 mm KCl, 0.75 mm MgCl 2 ), 200 M dntp, 2 pm of each primer, and 2 units of Taq DNA polymerase. The PCR had an initial denaturation at 94 C for 5 min, followed by 30 cycles of 1 min each. The initial annealing temperature of 65 C decreased 2 C in each cycle, alternating with temperatures of 94 C for 1 min, until a final annealing temperature of 55 C was reached, with a final extension at 72 C for 5 min. Reactions were performed in duplicate. Each set of reactions included a positive control. Additionally, positive controls were run for the reaction by adding 10 and 100 fg of purified DNA of T. cruzi and a negative control without the addition of DNA. The PCR product was visualized by electrophoresis in a polyacrylamide gel stained with silver. 22 RESULTS Parasitemia. All infected dogs gave a positive result for parasitemia upon the examination of fresh blood, which was characteristic of the acute phase of the disease after the first infection. Figure 1 shows the mean prepatent period (mean period between the day of inoculation and the detection of parasite in the blood after examination of fresh blood) and patent periods (mean number of days on which parasites are detected in the blood). After the first reinfection, the animals in groups C and D showed patent periods of two and 14.4

3 960 MACHADO AND OTHERS FIGURE 1. Curve of mean parasitemia in dogs infected/reinfected with strains and of Trypanosoma cruzi. days, respectively. The parasitemia level decreased dramatically with successive infections. The first reinfection levels were lower than those of the first infection (P 0.05); that of the second reinfection was negative on examination of fresh blood. Xenodiagnosis. There were no statistically significant differences in infection rates among the species of triatomines used in the xenodiagnosis, independently of the experimental group (P 0.05). Therefore, to assess the parasitemia by xenodiagnosis we used 40 nymphs/test, irrespective of the species. We observed that during the acute phase the infection rates of the triatomines reached 94.4% in the animals first infected with strain and 90% in those whose first infection was with the strain. Positive xenodiagnosis was observed up to the eighth, fifth, 23rd, and 15th month for groups A, B, C, and D, respectively (Figure 2). Among the dogs that were infected only once, all xenodiagnoses applied during the chronic phase of the disease were consistently negative from the ninth month onwards in the dogs in group A and from the sixth month onwards in those of group B (Figure 2). Serologic tests. Both the IIF and the ELISA detected high levels of IgG in the animals infected/reinfected by T. cruzi. The antibody titers detected in the reinfected dogs remained elevated, irrespective of the strain with which they were first infected, and presented similar profiles during the entire follow-up period (P 0.05). Among the dogs in groups A and B, (infected only once) the profiles were also similar (P 0.05). However, the antibody levels detected in these animals decreased over the months of observation, without ever becoming negative. The antibody levels detected during the FIGURE 2. Mean positivity of the xenodiagnoses of dogs infected once (Groups A and B) and those reinfected (Groups C and D) with strains and of Trypanosoma cruzi. The arrows indicate the time of reinfections; these are valid only for Groups C and D.

4 REINFECTION BY T. CRUZI IN DOGS 961 FIGURE 3. Profiles of the levels of IgG antibodies to Trypanosoma cruzi detected in dogs experimentally infected and/or reinfected with the parasite. A, indirect immunofluorescence test; B, enzyme-linked immunosorbent assay. chronic phase were greater in the dogs that had been reinoculated than in those infected only once (P 0.05) (Figure 3). Molecular characterization. Isoenzymatic profile. Samples of T. cruzi were isolated at different times during the entire follow-up period. All samples showed the zymodeme B profile corresponding to strain for all the enzymes studied, irrespective of the strain with which the animal was first infected and the triatomine species used in re-isolation. Variability of the mini-exon gene. Based on the mini-exon gene, the simultaneous presence of both strains was verified in three of the reinfected animals (Figure 4). The remaining reinfected animals presented the mini-exon gene profile of strain. Clinical evaluation. None of the dogs died of Chagas diseases during the study. The infected animals showed good general health, without significant alterations in body weight. A brief and oligosymptomatic acute phase was observed in all the animals infected and sporadic febrile episodes occurred during the first weeks after each inoculation. There were no signs of congestive cardiac insufficiency and/or electrocardiographic alterations during the entire follow-up. Necropsy. Macroscopically, visceromegalia was not seen in any of the necropsies. A whitish thickening in the form of plaque in the right auricle (chronic productive epicarditis) was identified in one of two dogs infected with strain and chronic productive perisplenitis was observed in a one animal infected with strain. Microscopically, myocarditis (Figure 5) was seen in the form of rare, small foci of mononuclear cells, with degenerative alterations and/or myocellular necrosis, in four dogs (1 in group A, 2 in C, and 1 in E). In three of these cases (1 in group A and 2 in C), there was also discrete chronic focal epicarditis. In one dog in group D, small foci of mononuclear cells were noted in

5 962 MACHADO AND OTHERS FIGURE 4. Polymerase chain reaction amplification products of mini-exon gene found in samples of Trypanosoma cruzi isolated from the infected/reinfected dogs (type basepairs; type basepairs). Lane 1, Group A; lane 2, Group B; lanes 3 and 4, Group C; lanes 5 and 6, Group D. The numbers on the left are the size markers used ( X 174 DNA digested with Hae III). NC negative control without DNA. the parietal endocardium. Rare, minuscule foci of fibrosis were identified in the myocardia of six dogs (1 each in groups A, B, and C and 3 in group D). No inflammatory foci were seen in the muscular layers or nervous intramural plexi of the digestive system (esophagus, stomach, and small and large intestines). Results of staining for nests of amastigotes with hematoxylin and eosin were negative in all organs, and in sections of heart stained with PAP. Discrete chronic epicarditis could be seen by microscopic examination in two noninfected dogs, and one showed focal periganglionitis in the myoenteric plexus of the esophagus. With regard to the remaining viscerae of both infected and noninfected animals, predominantly mononuclear inflammatory infiltrates were seen in the liver within the periportal spaces and occasionally around the efferent vein. Polymerase chain reaction. The PCR results for parasite DNA were negative in all fragments of heart tissue analyzed and were independent of the experimental group. DISCUSSION FIGURE 5. Myocarditis in the form of rare and small foci of mononuclear cells (A) and with degenerative alterations and/or myocellular necrosis (B) (magnification 400). Transmission of T. cruzi in endemic areas is intimately related to the density and index of intradomiciliary infection of triatomines and to the species of vector present. 25 Thus, individuals with prolonged exposure to these conditions in those areas are subject to reinfections by T. cruzi. Macedo and others 9 reported the case of an individual who lived in an endemic area and had the indeterminate form of the disease, subsequently dying of heart failure due to acute chagasic myocarditis. Therefore, these investigators suggested the possibility of human reinfection by T. cruzi. In the present study, which in part attempted to reproduce these possible reinfections, we observed that all dogs presented a discrete acute phase. The 34 months of the follow-up of the chronic phase were long enough to detect discreet clinical, electrocardiographic, and anatomo-pathologic alterations based on the classical studies of experimental infection in the dog. 13,14, 26,27 Clinically, the behavior of these animals always reflected the indeterminate chronic form of Chagas disease, even without the use of digestive radiology, which is consistent with the consensus in the literature. 28 The anatomo-pathologic examinations fully confirmed this clinical profile. 29,30 In general, after reinoculations with T. cruzi, the animals did not show the parasitemia levels characteristic of the acute phase of the disease. However, the profile of the parasitemia curve obtained was similar to that observed in acute Chagas disease. Brumpt 31 was the first investigator to demonstrate that animals infected with T. cruzi that survive an acute infection subsequently have a strong immunity to reinfection. In ex-

6 REINFECTION BY T. CRUZI IN DOGS 963 perimental studies performed on dogs, Costa and Costa 32 showed that chagasic infection conferred protection against to reinfections, concluding that it could be a result of a state of preimmunity or artificially acquired immunity. Nussenzweig and others 33 concluded that there was no difference in the protection of mice inoculated with two different strains of T. cruzi, suggesting that the defense mechanisms did not depend on antigenic differences between the strains. Recently, Lauria-Pires and Teixeira 8 concluded that reinfections with genetically characterized clones of T. cruzi did not aggravate morbidity and mortality in mice. Several investigators have shown consistently that acquired immunity confers partial protection on the animals against reinfections by the same or different strains. 5,33 35 We did not observe marked differences in the parasitemia profiles evaluated by xenodiagnoses during the acute phase. A gradual reduction of the parasitemia levels occurred until completely negative results were obtained for the examinations of animals that were not reinoculated. Parasitemias of long duration were only encountered in the animals that were reinoculated, in which the levels of parasitemia allowed up to 5.4% of the triatomines to be infected. Our findings confirm that reinfections may occasionally prolong the parasitemia, and that the immune system of the animals regulates the multiplication of the parasite when subjected to reinoculations. They also confirm that the second strain of T. cruzi may infect host without reproducing a characteristic acute phase The results of the xenodiagnoses observed in the dogs during the course of first infections and subsequent reinfections resemble those observed in humans. 38 The repeated examinations throughout the chronic phase show that the parasitemia profiles encountered may vary among individuals. Some of the animals presented sporadic or repeated positive results for parasites in their blood while others always remain negative. A study of the isoenzyme profiles of strains isolated from patients from the municipality of Bambui confirmed the simultaneous occurrence of two parasite populations in the same individual in 10% of the cases, which demonstrated the possibility of mixed infections occurring with different populations of T. cruzi (Romanha AJ, unpublished data). However, in our studies, only strain (ZB) was identified by isoenzymatic characterization of samples from dogs reinoculated with different strains. This was independent of the strain used in the initial infection. Although the passage of samples in culture was necessary to obtain enough parasites adequate for the preparation of enzyme extracts and DNA, we may have selected for one population during this procedure. Alternatively, the populations were present in the reinfected dogs, but with strain predominant. Both hypotheses could be explained in part by the identification of only strain by isoenzyme analysis. However, the miniexon gene assay, which seems to be more sensitive, showed the simultaneous presence of both parasite strains in three of the eight reinfected dogs. In this study, strain may have been selected in the majority of the reinfected dogs during passages in dogs and triatomines. These results reinforce the hypothesis that vertebrate and invertebrate hosts are capable of selecting different sub-populations of T. cruzi after infections with the same or different strains. Both the IIF and ELISA detected IgG antibodies in all infected dogs. The antibody levels increased considerably during the acute phase and remained elevated nearly throughout the entire follow-up. Antibody levels did not correlate either with the characteristics of the strain used in the initial infection or the clinical and histopathologic findings. Nevertheless, they were significantly higher in the dogs that received successive reinoculations. These data demonstrate that there is regulation of multiplication of the parasite through acquired immunity, which would explain the absence of circulating parasites even after the reinoculations, or their presence at levels only detectable by xenodiagnosis. Necroscopic examination of nine dogs infected with T. cruzi did not show macroscopic and/or microscopic alterations indicative of the cardiac and/or digestive forms of Chagas disease. In four (43%) of these infected animals, small, infrequent foci of myocarditis were detected by anatomo-pathologic examination, which is compatible with the indeterminate form of Chagas disease, and would be expected from the observed clinical data and electrocardiographs. These myocardial foci are similar to those described by Andrade and Andrade 14 and Andrade 39 in experimentally infected dogs. Lopes and others 40 also reported chronic focal carditis in five dogs naturally infected with T. cruzi. However, it should be emphasized that although the infiltrate consisted principally of plasmocytes in the cases described by Lopes and others, 40 this was not observed in our dogs, which were otherwise similar to those used by Andrade and Andrade, 14 and Andrade. 39 It should also be pointed out that both for our findings and those of the other investigators, elements are lacking that could attribute the etiology of Chagas disease with certainty to the inflammatory myocardial foci, since these did not contain T. cruzi. Nevertheless, the fact that the acute phase of the disease was confirmed in the infected dogs and no myocarditis was seen in noninfected animals indicates that these foci are related to Chagas disease. Analysis by the PCR for parasite DNA in the cardiac tissues gave negative results, despite the greater sensitivity of this technique. 41 Problems in the DNA extraction process or inhibition of the reaction can be ruled out since we used tissue fragments of uninfected dogs, to which were added 1.0 ng of T. cruzi DNA, as controls. Under these conditions, we observed amplification of parasite DNA. The PCR results corroborated those obtained by staining with hematoxylin and eosin and PAP, which did not show the presence of parasites in the tissues. The rare foci of fibrosis detected in six infected dogs probably represent myocarditis that may have occurred in the acute or chronic phases of T. cruzi infection. However, because of their rarity and small size, they did not cause clinical and/or electrocardiographic complications. These findings may be related to the absence of the parasite in this tissue as demonstrated by the results of staining with hematoxylin and eosin or PAP or the PCR. Discrete epicarditis was seen both in infected and noninfected animals. Therefore, it could not be considered specific for T. cruzi infection, as in humans. Our cardiac findings, similar to those of Andrade and An-

7 964 MACHADO AND OTHERS drade, 14 Andrade, 39 and Lopes and others, 40 differ from those reported by Pellegrino 26 and Lana and others, 15,42 who found diffuse fibrous cardiopathy and cardiac insufficiency in some infected dogs. However, Lana and others 15,42 worked with dogs of a different breed and another strain of the parasite and also inoculated a larger number of animals than were used in our study. These investigators also inoculated recently weaned puppies, attributed by Laranja 43 to be of great importance in the evolution of experimental Chagas disease. Similar observations were made by Dias 10 in relation to human Chagas disease. A predominantly mononuclear infiltrate was seen in the livers of dogs infected by T. cruzi, as well as in noninfected animals. Therefore, this observation must be nonspecific, as suggested by Lopes and others. 40 No macroscopic and/or microscopic alterations were observed in the esophagus and large intestine, organs frequently compromised by Chagas disease in humans, that could be attributed to this disease. This differs from the observations of megaesophagus and megacolon of Okumura and Corrêa-Neto. 13 Microscopic examinations of the only dog with periganglionitis in the myenteric plexus of the esophagus showed that it was not infected. Although this at first appears to be anomalous, this alteration is relatively frequent in healthy uninfected humans who died violently. 44 In conclusion, independent of the reinfections and the strain used in the initial infection, under the experimental conditions used we were unable to induce the cardiac and/ or digestive forms of chronic Chagas disease in dogs. Our findings show that all the animals, even those that were reinfected, continue to exhibit the indeterminate form of the disease. The results of this study should be analyzed taking into account other factors involved in the pathogenesis of Chagas disease, such as the number of parasites inoculated, the period of inoculum, and the intrinsic characteristics of the parasite strains and the host. Thus, the development of infection by T. cruzi in the dog resembles that in humans, so that within the limits of experimental design, it can be used as a model in studies of the natural history and immunopathology of Chagas disease. Acknowledgments: We thank Rosalida S. N. Lopes (Universidade Federal de Minas Gerais) and Urias A. Lamounier and Paulo A. Lamounier (Posto Avançado de Estudos Emmanuel Dias Fiocruz, Bambuí, Minas Gerais) for technical assistance. Financial support: This research was supported by Fundação de Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Oswaldo Cruz (FIOCRUZ). Authors addresses: Evandro M. M. Machado, Alexandre J. Fernandes, Silvane M. F. Murta, Alvaro J. Romanha, and João Carlos Pinto Dias, Centro de Pesquisas René Rachou, Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, Minas Gerais, Brazil. Ricardo W. A. Vitor, Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, Minas Gerais, Brazil, Sheila J. Adad, Deolino J. Camilo Júnior, Simone W. Pinheiro, and Edison Reis Lopes, Faculdade de Medicina do Triângulo Mineiro, Rua Getúlio Guaritá, 130, Uberaba, Minas Gerais, Brazil, Reprint requests: Evandro M. M. Machado, Centro de Pesquisas René Rachou, Fiocruz, Av. Augusto de Lima 1715, Belo Horizonte, Minas Gerais, Brazil, REFERENCES 1. 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